Probiotics offer significant health advantages as they enter the digestive system via diet or water intake, playing a crucial role in enhancing immunity, growth, gastrointestinal microbiota, and feed attribute. The main objective of study to focusing in the impact of Probiotic Functional Feed (PFF) on Nile tilapia (Oreochromis niloticus) exposed to challenges from Vibrio harveyi and Vibrio parahaemolyticus. The investigation aims to analyze the genes linked to immunity, hemato-biochemical indices, and the immunological response in tilapia. PFF is a vital component of fish feed production, providing suitable nutrition for various ages and stages to promote healthy growth. The study comprises five treatments: Negative Control (NC), Positive Control (PC), Rossellomorea marisflavi sp. DAS-SCF02, Agrococcus sp. RKDAS1, and a dual probiotic mix. A total of 150 Nile tilapia juveniles, weighing 2.56 ± 1.26 g, were administered PFF in triplicates. Significant improvements were observed in hematological indices, encompassing white blood cells (WBC), hemoglobin (Hb), red blood cells (RBC), hematocrit (Htc), and blood performance (BP) in probiotic-treated groups compared to control. Biochemical analysis revealed lower levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in fish fed with probiotics, accompanied by increased total protein, albumin, and globulin levels. Furthermore, probiotic-fed fish exhibited heightened blood glucose, total cholesterol, and triglyceride levels. Immunological assessments demonstrated increased lysozyme activity, intracellular superoxide anion production, reactive nitrogen species synthesis, and myeloperoxidase activity in probiotic-fed groups. Immune gene expression analysis revealed up-regulation of stress response, cytokine signaling, and immune defense-related genes (HSP70, IL-1β, I C3, IFN-α, IFN-γ, GF1, GH, IL-1, and Lyz). In a Vibrio challenge study, probiotic-fed fish exhibited improved survival rates, underscoring the protective effects of probiotics against bacterial infections. Overall, this research underscores the multifaceted benefits of probiotic supplementation in enhancing the health and immunity of tilapia.